Inflammatory response to retrotransposons drives tumor drug resistance that can be prevented by reverse transcriptase inhibitors

Significance The role of endogenous retrotransposons in cancer initiation and progression has been traditionally viewed in the context of their mutagenic activity. The main conclusion of the present work is that retrotransposons can stimulate treatment resistance by the induction of prosurvival inflammatory pathways, a mechanism that is distinct from retrotransposon-mediated mutagenesis. Furthermore, this mechanism is “druggable”, evidenced by partial reversion via reverse transcriptase inhibitors. In this regard, drugs targeting L1-expressing cells represent a category of anticancer agents that lack direct antitumor activity but are capable of suppressing tumor adaptability. This type of pharmaceutical is projected to improve the outcome of any anticancer treatment by extending progression-free survival following initial response.


Supplementary
. Tumor infiltrating lymphocytes abundances estimated from RNA sequencing. Absolute scores from CIBERSORTx for 25 mouse immune cell types are provided for samples from 4 different tumor types: primary tumor developed in untreated ('primary untreated') and in stavudine-treated ('primary STV') mice and recurrent tumors regrown following complete response to 17-DMAG treatment, in the presence and in the absence of stavudine in drinking water ('reccurent after DMAG' and 'Reccurent after DMAG+STV', correspondingly). For inference of immune cell composition of samples, we used CIBERSORTx online analysis platform (https://cibersortx.stanford.edu/) (1) was used to analyze gene expression profiles. Mouse-specific leukocyte signature matrix from ImmuCC (2)

Cytotoxicity Studies
Cells were plated at a density of 10,000 cells/well in 6-well plates and the treatment agents (1.5µM 17-DMAG, 250 ng/ml doxorubicin (Sigma) or combination of 1.5µM 17-DMAG and 10 µM STV or 250 ng/ml doxorubicin and 10 µM STV) were added. After 48h of treatment, cells were fixed using 1 mL per well staining solution (50% methanol, 50% water, 1% methylene blue dye). After incubation for 1h at room temperature, plates were rinsed three times with water and air-dried. 1% sodium dodecyl sulfate (SDS) solution was then added to the wells to solubilize the protein-bound dye and the optical density was measured using a plate reader (Perkin Elmer Victor 3x) in 96-well format.
Colony formation Assay 4T1 cells were passaged in the presence of 10 µM STV in parallel with untreated cells for four weeks. After this period colony formation assay was performed. Cells were seeded at a density of 10 6 cells per 10-cm Petri dish in triplicate. On the next day, cells were treated with 1.5 µM 17-DMAG. After 48h of 17-DMAG treatment, the medium was replaced with a fresh medium and the cells were cultured for another 5 days. On day seven, cells were fixed and stained with 1% methylene blue solution after washing with PBS. The number of colonies was counted. 4T1 tetL1(GLucAI) were passaged in three variants: untreated, with 400 ng/ml doxycycline (Doxy) (Sigma), and with combination of 400 ng/ml Doxy and 10 µM STV for four weeks. The concentrations of Doxy and STV used did not affect cell clonogenicity. The same experiment for DMAG-resistant cell variants was performed.
Quantitative RT-PCR 4T1 and 4T1R1 cells were seeded at a density of 10 6 cells per 10 cm Petri dish in duplicate. On the next day, half of the cells were treated with 1.5 µM 17-DMAG. After two hours of 17-DMAG treatment, cells were collected and total RNA was isolated using Trizol(Thermofisher Scientific, Fremont, CA) and then treated with DNase I (Sigma) following the manufacturer's protocol. Quantitative RT-PCR (qRT-PCR) was performed on a CFX384 Touch Real-Time PCR Detection System using the iTaq Universal One-Step Kit (BioRad) and 5 ng of RNA per reaction. Four replicate reactions were performed for each sample. GAPDH was used as a housekeeping gene for data normalization. Primer sequences are provided in Supplementary Table 2.

Immunostaining of cells and tumor sections
Cells were fixed for 15 min in 4% paraformaldehyde (PolySciences) in PBS (Corning) at 4°C, washed 3x in PBS, and incubated with blocking solution (5% normal donkey serum (Sigma), 0.2% triton x-100 (Sigma), PBS(Corning), 1% glycine (Sigma)) for 15 min. After fixation, cells were stained with primary antibodies against LINE-1 ORF1 (1:400 dilution, Millipore) or NF-κB p65 (1:200 dilution, Cell Signaling). Cells were then washed with 5 changes of PBS for 6-8h total at room temperature and stained overnight with secondary donkey anti-mouse antibody against whole molecule IgG conjugated with Cy3 (1:1000 dilution, Sigma) or AlexaFluor488 (1:1000 dilution, Thermofisher). Nuclei were counterstained with DAPI (Thermofisher). Samples were washed with several changes of PBS for a total of 4h, cleared, and mounted with ProLong Gold antifade reagent (Thermofisher). Images were collected with a Zeiss AxioImager 2 fluorescent microscope equipped with an AxioCam 702 digital camera using ZEN software 2.6 version. Tumors collected from in vivo study were dehydrated, embedded in paraffin, and sectioned into 5-10-micron sections using a microtome. The sections were gently placed on positively charged slides, deparaffinized, and rehydrated using xylene, ethanol, and double-distilled water. The sections were then boiled for 30 min in R-Universal antigen retrieval buffer (Electron Microscopy Sciences) for antigen unmasking and incubated in 3% hydrogen peroxide solution for 15 min. After blocking with 5% donkey serum (Sigma), sections were incubated overnight with primary antibodies for HER2 (1:200 dilution, DAKO), HSP70 (1:100 dilution, Cell Signaling), Ki67 (1:200 dilution, Thermo Scientific), Caspase 3 (Asp175) (1:100 dilution, Cell Signaling), Survivin (1:100 dilution, Cell Signaling). On the next day, the slides were washed and stained using the Ultravision ONE HRP polymer kit (Thermofisher) per the manufacturer's instructions. The sections were then counterstained with Mayer's hematoxylin (Sigma), dehydrated, mounted using Fisher Chemical Permount Mounting Medium (Thermofisher), and imaged using a Zeiss AxioImager 2 microscope equipped with an AxioCam 512 digital camera and ZEN software 2.6 version. Immunofluorescent staining for SMA was performed as described above except that after blocking with 5% donkey serum (Sigma), slides were incubated with a Cy3-conjugated mouse monoclonal antibody against SMA (1:1000 dilution, Sigma) for 30 min, washed and imaged.

RNA isolation
10-50 mg of frozen tissue samples, with the addition of 700 µl of Qiazol reagent, are first homogenized using Navy Rhino tubes in a Bullet Blender Homogenizer (Next Advance) for five minutes. The homogenate is then removed and incubated in a new tube at room temperature. After addition of chloroform, the homogenate is then separated into aqueous and organic phases by centrifugation. RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase. The upper, aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions for all RNA molecules from 18 nucleotides upwards. The sample is then applied to the miRNeasy Mini spin column, where the total RNA binds to the membrane and phenol and other contaminants are efficiently washed away. On-column DNAse digestion is also performed to remove any residual genomic DNA contamination followed by additional washes. High quality RNA is then eluted in 60 µl of RNase-free water.
Quantitative assessment of the purified total RNA is then accomplished by using a Qubit Broad Range RNA kit (Thermofisher), and concentration is determined by Ribogreen fluorescent binding to isolated RNA. The RNA is further evaluated qualitatively using RNA Nanotape on the 4200 Tapestation (Agilent technologies), where sizing of the RNA is determined, and a qualitative numerical score (RINe) is assigned.All RNA sequencing experiments were done in triplicates.

RNA-sequencing (RNA-seq)
Sequencing libraries were prepared with the RNA HyperPrep Kit with RiboErase (HMR) kit (Roche Sequencing Solutions) from 500ng total RNA. Following the manufacturer's instructions, the first step depletes rRNA from total RNA. The remaining RNA is DNase-digested to remove any genomic DNA contamination. Samples are then purified, fragmented, and primed for cDNA synthesis. Fragmented RNA is then reverse transcribed into first strand cDNA using random primers. The next step removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. Pure Beads (KAPA Biosystems) are used to separate the ds cDNA from the second strand reaction mix resulting in blunt-ended cDNA. A single 'A' nucleotide is then added to the 3' ends of the blunt fragments. Multiple indexing adapters, containing a single 'T' nucleotide on the 3' end of the adapter, are ligated to the ends of the doublestranded cDNA, preparing them for hybridization onto a flow cell. Adapter-ligated libraries are amplified by PCR, purified using Pure Beads, and validated for appropriate size on a 4200 TapeStation using D1000 Screentape (Agilent Technologies, Inc.). The DNA libraries are quantitated using the KAPA Biosystems qPCR kit (Sigma) and are pooled together in an equimolar fashion, following experimental design criteria. NovaSeq Standard-Each pool is denatured and diluted to 350pM with 1% PhiX control library (Illumina) added. The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge (Illumina), as determined by the number of sequencing cycles desired, and sequenced on a NovaSeq6000 instrument (Illumina) following the manufacturer's recommended protocol.